Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo


The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle–mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: In vitro delivery of Cas9 mRNA mediates efficient genome editing in cells.
Figure 2: In vivo delivery of Cas9 mRNA and AAV-HDR template cures type I tyrosinemia mice.
Figure 3: In vivo delivery of Cas9 mRNA and AAV corrects Fah mutation.

Accession codes

Primary accessions



  1. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  2. Doudna, J.A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

    Article  Google Scholar 

  3. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  Google Scholar 

  4. Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).

    Article  CAS  Google Scholar 

  5. Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    Article  CAS  Google Scholar 

  6. Aponte, J.L. et al. Point mutations in the murine fumarylacetoacetate hydrolase gene: animal models for the human genetic disorder hereditary tyrosinemia type 1. Proc. Natl. Acad. Sci. USA 98, 641–645 (2001).

    Article  CAS  Google Scholar 

  7. Azuma, H. et al. Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice. Nat. Biotechnol. 25, 903–910 (2007).

    Article  CAS  Google Scholar 

  8. Paulk, N.K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010).

    Article  CAS  Google Scholar 

  9. Schwank, G. et al. Functional repair of CFTR by CRISPR-Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013).

    Article  CAS  Google Scholar 

  10. Wu, Y. et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13, 659–662 (2013).

    Article  CAS  Google Scholar 

  11. Long, C. et al. Prevention of muscular dystrophy in mice by CRISPR-Cas9-mediated editing of germline DNA. Science 345, 1184–1188 (2014).

    Article  CAS  Google Scholar 

  12. Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  CAS  Google Scholar 

  13. Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102–106 (2015).

    Article  CAS  Google Scholar 

  14. Zuris, J.A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

    Article  CAS  Google Scholar 

  15. Chu, V.T., Weber, T. & Wefers, B. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).

    Article  CAS  Google Scholar 

  16. Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

    Article  CAS  Google Scholar 

  17. Khorsandi, S.E. et al. Minimally invasive and selective hydrodynamic gene therapy of liver segments in the pig and human. Cancer Gene Ther. 15, 225–230 (2008).

    Article  CAS  Google Scholar 

  18. Kay, M.A. State-of-the-art gene-based therapies: the road ahead. Nat. Rev. Genet. 12, 316–328 (2011).

    Article  CAS  Google Scholar 

  19. Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014).

    Article  CAS  Google Scholar 

  20. Love, K.T. et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl. Acad. Sci. USA 107, 1864–1869 (2010).

    Article  CAS  Google Scholar 

  21. Semple, S.C. et al. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 28, 172–176 (2010).

    Article  CAS  Google Scholar 

  22. Chen, D. et al. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J. Am. Chem. Soc. 134, 6948–6951 (2012).

    Article  CAS  Google Scholar 

  23. Kormann, M.S. et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 29, 154–157 (2011).

    Article  CAS  Google Scholar 

  24. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

    Article  CAS  Google Scholar 

  25. Kauffman, K.J. et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett. 15, 7300–7306 (2015).

    Article  CAS  Google Scholar 

  26. Sundararajan, S., Wakamiya, M., Behringer, R.R. & Rivera-Pérez, J.A. A fast and sensitive alternative for β-galactosidase detection in mouse embryos. Development 139, 4484–4490 (2012).

    Article  Google Scholar 

  27. Kim, H. et al. A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans. Genetics 197, 1069–1080 (2014).

    Article  Google Scholar 

  28. Barzel, A. et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature 517, 360–364 (2015).

    Article  CAS  Google Scholar 

  29. Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).

    Article  CAS  Google Scholar 

  30. Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2014).

    Article  Google Scholar 

  31. Mahiny, A.J. et al. In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency. Nat. Biotechnol. 33, 584–586 (2015).

    Article  CAS  Google Scholar 

  32. Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).

    Article  CAS  Google Scholar 

  33. Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011).

    Article  CAS  Google Scholar 

  34. Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).

    Article  CAS  Google Scholar 

  35. Chen, H.Z. et al. Canonical and atypical E2Fs regulate the mammalian endocycle. Nat. Cell Biol. 14, 1192–1202 (2012).

    Article  CAS  Google Scholar 

  36. Elliott, B., Richardson, C., Winderbaum, J., Nickoloff, J.A. & Jasin, M. Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cell. Biol. 18, 93–101 (1998).

    Article  CAS  Google Scholar 

  37. Goldberg, A.D. et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691 (2010).

    Article  CAS  Google Scholar 

  38. Findlay, G.M., Boyle, E.A., Hause, R.J., Klein, J.C. & Shendure, J. Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120–123 (2014).

    Article  CAS  Google Scholar 

  39. Cox, D.B., Platt, R.J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

    Article  CAS  Google Scholar 

  40. Atabai, K. et al. Mfge8 is critical for mammary gland remodeling during involution. Mol. Biol. Cell 16, 5528–5537 (2005).

    Article  CAS  Google Scholar 

  41. Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  Google Scholar 

  42. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  Google Scholar 

  43. Bolukbasi, M.F. et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat. Methods 12, 1150–1156 (2015).

    Article  CAS  Google Scholar 

  44. Zhu, L.J. et al. ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinformatics 11, 237 (2010).

    Article  Google Scholar 

  45. Zhu, L. Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology. Front. Biol. 10, 289–296 (2015).

    Article  CAS  Google Scholar 

  46. Zhu, L.J., Holmes, B.R., Aronin, N. & Brodsky, M.H. CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems. PLoS One 9, e108424 (2014).

    Article  Google Scholar 

Download references


We thank M. Grompe, S. Levine, T. Jacks, P. Sharp, E. Sontheimer, C. Mello, P. Zamore, M. Moore, T. Flotte, T. Tammela, F. Sanchez-Rivera, T. Papagiannakopoulos, D. Wang, J. Moore and A. Vegas for discussions and for sharing reagents, S. Hough for technical assistance and K. Cormier for histology. This work is supported by grants from the National Institutes of Health (NIH), 5R00CA169512 and Worcester Foundation (to W.X.). H.Y. is supported by Skoltech Center and 5-U54-CA151884-04 (NIH Centers for Cancer Nanotechnology Excellence and the Harvard-MIT Center of Cancer Nanotechnology Excellence). Y.D. acknowledges support from the National Institute of Biomedical Imaging and Bioengineering for his postdoctoral fellowship 1F32EB017625. V.K. acknowledges support from the Russian scientific fund, grant number 14-34–00017. This work is supported in part by Cancer Center Support (core) grant P30-CA14051 from the NIH. We thank the Swanson Biotechnology Center for technical support. We thank C. Wang at Boston Children's Hospital Viral Core for AAV prep (supported by core grant 5P30EY012196-17). The authors acknowledge the service to the MIT community of the late S. Collier.

Author information

Authors and Affiliations



H.Y., W.X. and D.G.A. designed the study. H.Y. and W.X. directed the project. H.Y., C.-Q.S., J.R.D., L.J.Z., Y.L., Q.W., J.Y., S.S., A.B., A.G., M.F.B., A.P., S.W. and R.L.B. performed experiments and analyzed data. G.G., Z.W., Y.D., V.K., S.A.W. and R.L. provided reagents and conceptual advice. H.Y., W.X. and D.G.A. wrote the manuscript with comments from all authors.

Corresponding authors

Correspondence to Wen Xue or Daniel G Anderson.

Ethics declarations

Competing interests

D.G.A., H.Y., J.R.K. and W.X. have applied for patents on the subject matter of this paper. D.G.A. is a scientific co-founder of CRISPR Therapeutics.

Integrated supplementary information

Supplementary Figure 1 Cas9 mRNA nanoparticles characterization.

(a) nano.Cas9 formulation scheme. Cas9 mRNA was mixed with C12-200, DOPE, Cholesterol, C14PEG2000 and arachidonic acid in a microfluidic chamber. (b) nano.Cas9 structure is characterized by cryo-TEM. Scale bar indicates 100nm. (c) Average diameter of nano.Cas9 was measured by dynamic light scattering. The size of nano.Cas9 (d) and the polydispersity index (PDI) (e) were measured 0, 7, 11 or 18 days after formulation and storage at 4˚C.

Supplementary Figure 2 The expression of proteins in mouse liver after mRNA nanoparticles treatment.

(a) C57bl/6 mice were i.v. injected with nanoparticles encapsulated with β-gal (b and c) or Cas9 mRNA (nano.Cas9, d and e), and livers taken. (b) The expression of β-gal protein was measured in liver lysate at 14 hours after injection. (c) The activity of β-gal in liver sections was determined by salmon-gal assay. Scale bar indicates 200 µm. (d) The expression of Cas9 protein was measured in liver lysate 14 hours after injection. 50μg negative control samples mixed with 10, 1 or 0.1ng Cas9 protein served as positive controls. β-actin served as a loading control in (b) and (d). (e) The Cas9 mRNA level in liver was determined by qRT-PCR at 4, 14, and 24 hours after injection (n=3 mice).

Supplementary Figure 3 Cas9 mRNA nanoparticles are well tolerated.

C57/Bl6 mice were treated with 2mg/kg nano.Cas9, and histology (a), the levels of liver damage markers (b) and plasma cytokines (c) were determined after 24 hours. Scale bar indicates 50μM. (n=4 mice).

Supplementary Figure 4 The time course of sgRNA expression in mouse liver.

Mice were injected with AAV-HDR and livers taken at 0, 3, 7 and 14 days after injection. qRT-PCR was performed to determine sgRNA expression in liver. The expression levels were normalized to Day 3 (n = 4 mice).

Supplementary Figure 5 A PCR approach proves substitution of the correct sequence.

(a) Design of the PCR primers. Blue arrow indicates the reverse PCR primer, which is outside the repair template. The sequence of the forward primer is presented, and “G” and “CC” in the corrected sequence are highlighted. (b) Genomic DNA of the liver tissue was extracted, and PCR was performed using the primers in (a). The predicted size of PCR product is 1.02kb. A representative sample from each group is shown (n = 3 mice). (c) The PCR product from (b) was cloned to a TA cloning vector and Sanger sequenced. The corrected “G” and “CC” are highlighted.

Supplementary Figure 6 Viral delivery of Cas9 does not increase HDR rate compared to mRNA delivery.

(a) Design of AAV-HDR template. Four “G” point mutations resulting in stabilization of β-Catenin are highlighted. Ad.Cas9 is an adenovirus expressing Cas9. (b) β-Catenin IHC. AAV-HDR-Ctnnb1 alone serves as a control. Arrows denote β-Catenin positive hepatocytes. (c-d) The Ctnnb1 locus in the liver total DNA of Ad.Cas9+ AAV-HDR-Ctnnb1 treated mice (n=2) were deep sequenced to measure indels. (e) β-Catenin positive hepatocytes were counted to determine the percentage of HDR. P < 0.01 (n = 3 mice). mRNA delivery of Cas9 yields higher rate of HDR for Fah (>6%).

Supplementary Figure 7 Cas9 mRNA delivery has minimal off-target effects at assayed sites in vivo.

(a) Top ranking off-target sites (OT1, OT3 and OT4) for sgFah and the predicted score (Hsu et al, 2013). Mismatch bases are in red. Score for the wildtype sgFah.2 targets the mutant Fah which has one mismatch with wildtype Fah (wt Fah). (b) Indel frequency is low and is comparable between control mouse and nano-Cas9+ AAV-HDR mouse. OT1, OT3 and OT4 regions were PCR amplified from mouse liver genomic DNA and analyzed by deep sequencing. (c) Surveyor assay did not detect indels at OT1, OT3 and OT4. Predicted size of uncut and cut bands are indicated.

Supplementary Figure 8 Indel rate measured by deep sequencing for GUIDE-Seq off-target sites.

OT1 is the strongest off-target sites identified by GUIDE-Seq. GOT1-11 are additional genomic sites that displayed GUIDE-Seq oligonucleotide insertions. (a) Mouse Hepa1-6 liver cells transfected with pX330.sgFah.2. #1 and #2 are replicates. (b) Mouse livers treated with nano.Cas9 and AAV-HDR (treated) or control treated (control). Fah is the on-target site. See Table S9 for details.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–6 and Supplementary Sequences (PDF 3895 kb)

Supplementary Table 7

sgRNA2 GUIDE-seq +&- strand peaks (PDF 40329 kb)

Supplementary Table 8

sgRNA2 GUIDE-seq merged peaks (PDF 75 kb)

Supplementary Table 9

Deep sequencing of off-target sites (PDF 192 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yin, H., Song, CQ., Dorkin, J. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol 34, 328–333 (2016).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research